Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression

Significance The Wnt signaling pathway, often deregulated in cancer and other diseases, remains poorly understood in lipid metabolism, particularly lipid mobilization. This study reveals that active Wnt/Wg signaling potently reduces lipid accumulation in Drosophila adipocytes by boosting lipolysis and inhibiting lipogenesis and fatty acid β-oxidation. Key Wnt target genes controlling fat storage and mobilization were identified; active Wnt signaling suppresses their transcription, while reduced Wnt signaling enhances it, thereby modulating triglyceride metabolism. Furthermore, active Wnt signaling directly represses the transcription of these lipid metabolism-related genes. These findings suggest that Wnt signaling-induced transcriptional repression regulates lipid homeostasis, balancing triglyceride storage and mobilization in Drosophila adipocytes.


This PDF file includes:
Supplementary Materials and Methods   Supplementary Materials and Methods: Drosophila stocks and maintenance: The flies were maintained at 25˚C and fed with a standard diet comprising cornmeal, molasses, and yeast medium.Our analyses in this study focused exclusively on female larvae in the third instar wandering stage.Details about the specific strains and their genotypes are provided in Table S1.Arm EGFP and Nkd EGFP are intronic insertion lines with EGFP tags, generated using the MiMIC (Minos Mediated Integration Cassette) gene trap vector (1).The specific genotypes are 'Mi{PT-GFSTF.1}armMI08675-GFSTF. 1 ' and 'Mi{PT-GFSTF.1}nkdMI00209-GFSTF.1 /TM3, Sb 1 Ser 1 ', respectively.

Cell biological analyses:
The methods for generating somatic clones in the larval fat body, dissection, and fixation of the larval fat body, as well as the protocols for immunostaining or staining with BODIPY, LipidTox, DAPI, and phalloidin, have been previously described (2,3).
Quantification of TG levels involved thin layer chromatography (TLC) and a TG quantification colorimetric kit, following established procedures as described earlier (2,3).Additionally, for both S2R+ cell pellets or whole larvae, total lipids were extracted and applied to the TLC lanes.
Lipid levels were normalized with respect to the total protein levels in each corresponding sample.
Immunoblotting: Cultured S2R+ cells were rinsed and suspended in ice-cold 1×PBS, followed by cell lysis in Tropix Lysis Solution (Applied Biosystems #2005117) containing a protease inhibitor cocktail (Roche #11836170001).This lysate was incubated on ice for 15 minutes, then subjected to centrifugation at 16,000×g for 30 min at 4°C.Protein concentrations were determined using Quick Start™ Bradford 1x Dye Reagent (Bio-Rad #5000205).For each sample, 20 μg of total protein was loaded into 10% Mini-PROTEAN TGX Gels (Bio-Rad #4561034) in SDS running buffer.The proteins were transferred onto nitrocellulose membranes using the Trans-Blot Turbo RTA Mini 0.2 µm Nitrocellulose Transfer Kit (Bio-Rad #1704270).
The membranes were then incubated with a 1:1000 dilution of the anti-Arm primary antibody from the Developmental Studies Hybridoma Bank (DSHB Hybridoma Product N2 7A1 Armadillo, deposited by Eric Wieschaus), and a 1:2000 dilution of anti-Actin (Invitrogen #MA5-11869).A secondary antibody, anti-mouse-HRP-conjugated (Jackson ImmunoResearch), was used at a 1:5000 dilution.Visualization of the results was performed using the Western Lightning Plus-ECL (Perkin Elmer LLC #NEL105001EA).
The HCR RNA-FISH assay: The multiplexed in situ HCR RNA-FISH assay was performed as described previously (3), using specific probe sets and amplifiers obtained from Molecular Instruments.For probe sets targeting specific genes and their respective amplifiers, the following combinations were used : B1-Alexa Fluor 488 amplifiers with probe sets for FASN1 (lot number PRQ415), fz3 (RTD102), and Lsd-1 (PRQ418); B2-Alexa Fluor 594 amplifiers with the probe sets for AcCoAS (lot number PRQ416) and Lsd-2 (PRQ419); and B3-Alexa Fluor 647 amplifiers with probe sets for ACC (lot number PRQ417), Acox57D-d (PRQ420), CRAT (RTD104), and Hnf4 (PRR571).Confocal images were captured using a Zeiss LSM900 confocal microscope system.Subsequently, the obtained images were processed using Adobe Photoshop 2021.
For dual isotope radiolabeling experiments, S2R+ cells were seeded in six-well plates (2 ml/well; 3.5x10 6 cells/ml) for 24h.Radioactively labeled [ 14 C(U)]D-glucose (5 µl/well; 6.0×10 8 dpm/µmol) and [9,10-3 H(N)] Palmitic acid (2 µl/well; 1.05×10 11 dpm/µmol) were added to the cells.Next, cells were treated with either WCM or CM.For WCM preparation, S2-Tub-wg cells were seeded 24 h before collecting the supernatant (5x10 6 cells/ml) by centrifuging.Control medium (CM) was prepared using S2R+ cells.S2R+ cells were collected from CM and WCMtreated wells at 0hr, 12hr, and 24hr.Cell pellets were washed with 1x PBS and TLC was performed as previously described (2,4), with the following minor modifications.Specific regions corresponding to FFAs and TGs were scraped from the TLC plates and collected into scintillation vials.Counts per minute (cpm) values for 3 H and 14 C were determined by counting in 4 ml of CytoScint™ Liquid Scintillation Cocktail (Catalog number: 882453) in a Hidex 300 SL™ automatic liquid scintillation counter.Dpm (disintegrations per minute) values for 3 H and standards.Efficiencies of 3 H and 14 C counting were 33.06% and 87.23% respectively for an optimized period of 100 seconds measuring time.Dpm values for 14 C and 3 H within TG and FFA were calculated using the respective efficiency figures. 14C/ 3 H ratios were calculated, and normalized ratios were plotted.This protocol was used to perform dual isotope radiolabeling experiments to quantify TGs and FFAs in the cells over time to WCM treatment compared to the control.

RNA-seq sample preparation, library preparation, sequencing, differential gene expression
analysis, and gene ontology enrichment analysis: Total RNA was extracted from dissected fat bodies of 20 third-instar larvae for each biological replicate.TRIzol Regent (Invitrogen) and the Quick-RNA MiniPrep kit (Zymo Research, R1055) were used for the RNA extraction and purification following the manufacturer's instructions.Ribosome-depleted stranded RNA libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, 20020596) following to the manufacturer's manual.The prepared RNA libraries were sequenced by the BGI Americas Corporation.Sequencing reads were aligned to the Dm6 reference genome using the RNASTAR aligner (5).A count table was generated using featureCounts from Subread (6).Differential gene analysis within each group of experiments was performed by DESeq2 (7).
Genes with adjusted p-value < 0.05 were considered differentially expressed.Gene ontology enrichment analysis was carried out using ClusterProfiler and DAVID GO (8,9).The results of the gene ontology enrichment analysis were visualized using ggplot2 (10).Heatmaps were generated for visualization using the complexHeatmap package (11).
Quantitative proteomics analysis: Total proteins extracted from third instar whole larvae with the following genotypes: Axn 127 homozygous mutant and w 1118 (used as the control) were treated with 2.0 µM of BTZ mixed in fly food or DMSO (used as the control) (2).The samples were then analyzed using quantitative proteomics.Protein digestion was conducted using the filteraided proteome preparation (FASP) with slight modifications (12).Briefly, proteins were reduced with 100 mM DTT at 37 °C for 1 h, and the lysates were transferred to the Microcon YM-30 centrifugal filter units (EMD Millipore Corporation, Billerica, MA).The lysis buffer was replaced twice with 200ul UA (8M Urea, 100mM Tris.Cl pH8.5).Alkylation was performed using 55 mM iodoacetamide (IAA, Sigma-Aldrich, Saint Louis, MO), the denaturing buffer was replaced with a buffer containing 0.1 M triethylammonium bicarbonate (TEAB, Sigma-Aldrich).
Proteins were then digested using sequencing grade trypsin (Promega, Madison, WI) at 37 °C overnight.The resulting tryptic peptides were labeled with acetonitrile-dissolved TMT reagents (Thermo Scientific, Rockford, IL).Equal amounts of labeled samples were mixed, and prefractionation was carried out using reversed-phase (RP)-high-performance liquid chromatography (HPLC).Peptides were fractionated on a phenomenex gemini-NX 5u C18 column (250 x 3.0 mm, 110 Å) (Torrance, CA, USA) employing a Waters e2695 separations HPLC system.The separated samples were collected and combined into 10 fractions.All fractions were dried using a Speed-Vac concentrator and subsequently desalted by StageTip (13).
LC-MS/MS analysis was performed using an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) coupled online to an Easy-nLC 1000 in the data-dependent mode.Peptides were separated on a capillary analytic column (length: 25 cm, inner diameter: 75 μm) packed with C18 particles (diameter: 5 μm).The analysis was conducted in positive ion mode, and spectra were acquired within the mass range of 300-1800 m/z.Higher-energy collisional dissociation (HCD) was used to fragment the fifteen most intense ions from each MS scan.The raw MS files were processed through a database search using MaxQuant software (version 1.5).For this search, the Drosophila melanogaster proteome sequence database downloaded from uniprot (https://www.uniprot.org/)was used.The search parameters were configured as follows: type of search: MS2; protease used for protein digestion: trypsin; type of isobaric labels: 6-plex TMT; minimum score for unmodified peptides: 15; default values were applied for all other parameters.
Lipid sample preparation and shotgun lipidomics analysis: Larval fat bodies were dissected and promptly frozen using liquid nitrogen.Whole larvae samples were briefly rinsed in PBS and then frozen in liquid nitrogen.The frozen tissues were pulverized using a mortar and pestle precooled with liquid nitrogen.The powdered samples were further homogenized by suspending them in a 10-fold diluted PBS using a Precellys Evolution ® Homogenizer, running at 6000 rpm for 20 seconds, followed by 10-second pause.This homogenization cycle was repeated three times consecutively at 4 °C.Individual homogenates were subject to a protein assay to determine their protein content.An aliquot of each homogenate was then transferred to disposable glass test tubes.To enable quantification of various lipid classes, a mixture of lipid internal standards was added to each test tube, the quantities adjusted based on the tissue's protein content.Lipid extraction was performed by a modified Bligh and Dyer method, as previously described (14).
Following extraction, all lipid extracts were flushed with N2, capped, and stored at -20 ºC for future analysis.
For the ESI-MS analysis conducted after direct infusion, individual lipid extracts were further diluted to reach a final concentration of approximately 500 fmol/µL.The mass spectrometric analysis for lipids was conducted using a QqQ mass spectrometer (Thermo TSQ Altis, San Jose, CA).This instrument was equipped with an automated nanospray device (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY), which facilitated the ionization of lipid species through nano-ESI.This instrument was operated using Xcalibur software as previously described (15).To identify and quantify all lipid molecular species of interest, an inhouse automated software program was used, adhering to the principles for quantification by mass spectrometry as elaborated previously (15).The fatty acyl chains of lipids were identified and quantified through neutral loss scans or precursor ion scans of corresponding acyl chains.These calculations were also performed using the same in-house software program (15).To ensure accurate quantification and comparisons, all lipid measurements were normalized to the protein content of the respective samples.CRISPR Cas9 mediated tagging of dTCF/Pan with EGFP: dTCF gene structure information was retrieved from FlyBase (FBgn0085432) and primary amino acid sequence was retrieved from Uniprot (Q8IMA8).We analyzed the domain structure of the protein (https://www.ebi.ac.uk/interpro/result/InterProScan/ iprscan5-R20220926-005559-0039-31654548-p1m/).Based on these analyses, we decided to introduce the EGFP tag at the C-terminus of the dTCF protein.Using the CRISPR Optimal target finder tool (http://targetfinder.flycrispr.neuro.brown.edu),two suitable sgRNAs target sites were strategically located within the last intron and 3'-UTR regions of the dTCF gene.The selected sgRNAs were then cloned into the pCFD3 vector, as described by Port et al.
(http://www.crisprflydesign.org/plasmids/)(16).Primer design for subsequent steps was accomplished using NEB Builder (https://nebuilder.neb.com/#!/), and primer sequences are provided in Table S2.For actual gene editing and tagging, we employed the NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520).This kit facilitated the introduction of both upstream and downstream homology arms, each spanning 1000bp each, as well as the last intronexon regions and the EGFP coding region into a donor vector.The BAC clone 'BACR21B19', obtained from the BACPAC Resources Center, was used as the template for PCR to generate necessary DNA fragments.The donor vector and two sgRNA constructs in PCDF3 were injected into embryos, and the screening of the transgenic flies carrying endogenous EGFP tag was carried out using classical fly genetic techniques.Additionally, PCR validation using genomic DNA extraction and sequencing was conducted to verify the successful integration of the EGFP tag.
CUT&RUN sequencing to identify the genome-wide dTCF-binding sites: For each sample, 20 wing discs from third-instar wandering larvae were dissected in Schneider medium.The wing discs were then transferred into Eppendorf tubes containing 100 µL 1x Wash buffer (from the CUT&RUN assay kit #86652, CST).We added 10 µL of activated Concanavalin A Magnetic bead suspension and allowed the tube to rotate at room temperature for 10 min.The Eppendorf tubes were placed on a Magnetic rack, the wash buffer was removed, and the beads were resuspended by adding 100 µL DBE buffer (provided in the kit).This resuspension step was conducted for 10 min at room temperature.We added 100 µL Antibody binding buffer containing the appropriate amount of primary antibody (anti-GFP, ab290) or negative control (rabbit mAb IgG from the kit).These tubes were placed on a nutator and incubated overnight at 4 °C.The subsequent steps were carried out by following the manufacturer's instructions as provided in the kit.The extracted DNA was purified by using DNA purification buffers and spin columns (#14209S, CST).DNA libraries were constructed using the TruSeq ChIP SMP Prep kit (IP-202-1012, 15034288, Illumina), following the sample preparation guide.
The sequencing data underwent processing using nf-core/cutandrun v2.0, an integrated pipeline designed for the CUT&RUN assay (17).The alignments from the pipeline underwent additional processing for peak calling with a significance threshold of -q 0.0001 and subsequent normalization using MACS3, which can be accessed at https://macs3-project.github.io/MACS/.
For anti-GFP samples, the alignments were provided through the "-t" option, while control (IgG) samples utilized the "-c" option.To visualize the results in IGV (Integrative Genomics Viewer) with the Drosophila genome (assembly Release dm6) as the reference, we transformed the averaged and normalized pileup bedgraph file, which was generated alongside the peak calling step through MACS3 "-B" option, into a bigwig file.This conversion was accomplished using the bedGraphicToBigWig tool from the UCSC toolkit.The analysis of dTCF binding profiles was conducted using deepTools.To identify motifs within dTCF-binding sites, we utilized the narrow peaks identified from above MACS3 output.These peaks were then intersected with the regions ±2000bp around genes showing alterations in both Axn RNAi and slmb RNAi RNA-seq data, by the intersect function from bedtools (18).The intersected peaks sequences were obtained by getfasta function from bedtools.For de novo motif discovery, we input these sequences to the xstreme function from the MEME-Suite package with default settings (19).

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analyses: ChIP analyses
were performed using dTCF/Pan EGFP or w 1118 (control) embryos collected over 24 hours, following a published protocol (20) with minor adjustments.After fixation and quenching of cross-linking, chromatin was fragmented to sizes ranging from 100 to 300 bp using a Covaris S220 Focused Ultrasonicator.The fragmented chromatin was then incubated overnight at 4˚C with GFP-Trap coupled to magnetic agarose beads (Bulldog, GTMA-020), while binding control magnetic agarose (Bulldog, BMAB-020) was used as a negative control.After washing, the chromatin was eluted from the beads, and the DNA was decrosslinked from protein.Finally, qPCR was performed using the purified DNA as the template, with specific primers listed in Table S3.S1 to S3: Table S1 List of the Drosophila stocks used in this study.

Figure
Figure legends for Supplementary Figures S1 to S16

Figures
Figures S1 to S16

Tables
Figure legends for Supplementary Figures S1 to S16:

Fig. S4
Fig. S4 Bioinformatic analyses of RNA-seq data for genes altered in Axn RNAi or slmb RNAi fat bodies.(A) Heatmap illustrating gene expression changes in fat bodies related to the 'Wnt signaling' pathway.(B, C) Pathway enrichment bubble plots displaying major pathways significantly altered in larval fat body upon depletion of either Axn (B) or slmb (C).Genotypes: (B) SREBP-Gal4/UAS-Axn RNAi ; +; and (C) SREBP-Gal4/+; UAS-slmb RNAi /+.(D) Heatmap displaying downregulation of gene expression in the 'Biosynthesis of unsaturated fatty acids' pathway in Axn RNAi or slmb RNAi fat body.(E) Heatmap illustrating changes in gene expression for genes related to the 'Peroxisome' pathway.

Fig. S11 .
Fig. S11.Generation and validation of the dTCF EGFP Drosophila strain.This figure outlines the process of creating and validating the dTCF EGFP Drosophila strain.(A) The design of the donor template using the pGEM-T-dTCF EGFP vector.(B) The validation of the dTCF EGFP line by PCR using genomic DNA from dTCF EGFP homozygous larvae.(C) Sequencing results confirming the presence and accuracy of the dTCF EGFP in the genomic DNA.(D/D') Localization of dTCF in the nuclei of larval adipocytes.(E/E') Localization of dTCF in the nuclei of wing imaginal disc cells.(D'/E') Merged images with DAPI staining of nuclei (blue).The scale bar in panel D' applies to images D/D' and E/E': 10 μm.Fig.S12.Visualization of dTCF binding at genomic loci in CUT&RUN assay data from wing discs and purified adipocyte nuclei samples.To facilitate comparison, the tracks are

Fig. S13 .
Fig. S13.Motif enrichment analyses of genes with called dTCF/Pan binding peaks.(A, B) Identification of a dTCF/Pan motif based on motif enrichment analysis of upregulated genes in both Axn RNAi and slmb RNAi adipocytes (A), and all called peaks in genes altered in both Axn RNAi and slmb RNAi adipocytes (B).For comparison, screenshots of motif bound by TCF HMG domain (C; from PMID 24516405) and LEF2 motif (D; from PMID 20696899) are included.(E, F) Validation of the CUT&RUN results using the ChIP-qPCR assay.(E) Relative enrichment of dTCF EGFP was assessed on the promoters of Wnt target gene nkd, and lipid metabolism-related genes, FASN1 and Lsd-1.Samples were from dTCF EGFP homozygous embryos, with the IgG control shown in black.The primer pair 'nkd-1C' was positioned away from the dTCF EGFPbinding peaks ('nkd-2C') in the nkd promoter region.(F) Negative controls used w 1118 embryos.

Fig. S14 .
Fig. S14.Pathway enrichment analyses of genes altered in both Axn RNAi and slmb RNAi adipocytes with called dTCF/Pan binding peaks.This analysis identified the Wnt signaling pathway and several pathways related to lipid metabolism, which are highlighted.

Fig. S15 .
Fig. S15.dTCF/Pan binding at diverse genomic loci.This figure provides a visual representation of dTCF binding at various genomic loci, organized by their functional pathways.Genes activated by Wnt signaling are shown in red tracks, while genes downregulated by Wnt signaling are displayed in blue tracks.(A) Genes related to the Wnt signaling pathway.(B) Genes related to fatty acid biosynthesis pathway.(C) Genes related to LDAPs and lipid droplets.The asterisk (*) indicates the transcription start site (TSS).The y-axis is autoscaled, while different transcript isoforms are consolidated and displayed in magenta beneath each respective track.Fig. S16.dTCF/Pan binding at diverse genomic loci.(A) Genes related to FAO and the electron transport chain.(B) Genes related to peroxisome.Genes downregulated by Wnt

D
Fig. S4 Bioinformatic analyses of RNA-seq data for genes altered in Axn RNAi or slmb RNAi fat bodies.(A) Heatmap illustrating gene expression changes in fat bodies related to the 'Wnt signaling' pathway.(B, C) Pathway enrichment bubble plots displaying major pathways significantly altered in larval fat body upon depletion of either Axn (B) or slmb (C).Genotypes: (B) SREBP-Gal4/UAS-Axn RNAi ; +; and (C) SREBP-Gal4/+; UAS-slmb RNAi /+.(D) Heatmap displaying downregulation of gene expression in the 'Biosynthesis of unsaturated fatty acids' pathway in Axn RNAi or slmb RNAi fat body.(E) Heatmap illustrating changes in gene expression for genes related to the 'Peroxisome' pathway.

Fig. S13 .
Fig. S13.Motif enrichment analyses of genes with called dTCF/Pan binding peaks.(A, B) Identification of a dTCF/Pan motif based on motif enrichment analysis of upregulated genes in both Axn RNAi and slmb RNAi adipocytes (A), and all called peaks in genes altered in both Axn RNAi and slmb RNAi adipocytes (B).For comparison, screenshots of motif bound by TCF HMG domain (C; from PMID 24516405) and LEF2 motif (D; from PMID 20696899) are included.(E, F) Validation of the CUT&RUN results using the ChIP-qPCR assay.(E) Relative enrichment of dTCF EGFP was assessed on the promoters of Wnt target gene nkd, and lipid metabolism-related genes, FASN1 and Lsd-1.Samples were from dTCF EGFP homozygous embryos, with the IgG control shown in black.The primer pair 'nkd-1C' was positioned away from the dTCF EGFP -binding peaks ('nkd-2C') in the nkd promoter region.(F) Negative controls used w 1118 embryos.
Fig. S14.Pathway enrichment analyses of genes altered in both Axn RNAi and slmb RNAi adipocytes with called dTCF/Pan binding peaks.This analysis identified the Wnt signaling pathway and several pathways related to lipid metabolism, which are highlighted.

C
Fig. S15.dTCF/Pan binding at diverse genomic loci.This figure provides a visual representation of dTCF binding at various genomic loci, organized by their functional pathways.Genes activated by Wnt signaling are shown in red tracks, while genes downregulated by Wnt signaling are displayed in blue tracks.(A) Genes related to the Wnt signaling pathway.(B) Genes related to fatty acid biosynthesis pathway.(C) Genes related to LDAPs and lipid droplets.The asterisk (*) indicates the transcription start site (TSS).The y-axis is autoscaled, while different transcript isoforms are consolidated and displayed in magenta beneath each respective track.

Fig. S16 .
Fig. S16.dTCF/Pan binding at diverse genomic loci.(A) Genes related to FAO and the electron transport chain.(B) Genes related to peroxisome.Genes downregulated by Wnt signaling are displayed in blue tracks.The y-axis is autoscaled, while different transcript isoforms are consolidated and displayed in magenta beneath each respective track.

Visualization of dTCF binding at genomic loci in CUT&RUN assay data from wing discs and purified adipocyte nuclei samples.
To facilitate comparison, the tracks are color-coded as follows: blue tracks represent genes downregulated by Wnt signaling in the wing disc sample, red tracks indicate genes stimulated by Wnt signaling in the wing disc sample, and orange tracks depict data from the purified adipocyte nuclei sample.Arrows highlight overlapping peaks observed in both sample sources, with a noticeable increase in background noise peaks in the purified adipocyte nuclei sample.The y-axis is autoscaled, while multiple transcript isoforms are collapsed and presented in magenta beneath each respective track.Specific genes included are Hnf4 (A), FASN1

Table S1 List of the Drosophila stocks used in this study
a kind gift from Dr. Yashi Ahmed